The present invention relates generally to compositions and methods useful for the study of cascades leading to the activation of nuclear factor xcexaB (NF-xcexaB) and for treating diseases associated with such pathways. The invention is more particularly related to a stimulus-inducible IxcexaB kinase (IKK) signalsome, component IxcexaB kinases and variants of such kinases. The present invention is also related to the use of a stimulus-inducible IKK signalsome or IxcexaB kinase to identify antibodies and other agents that inhibit or activate signal transduction via the NF-xcexaB pathway.
Transcription factors of the NFxcexaB/Rel family are critical regulators of genes involved in inflammation, cell proliferation and apoptosis (for reviews, see Verma et al., Genes Dev. 9:2723-35, 1995; Siebenlist, Biochim. Biophys. Acta 1332:7-13, 1997; Baeuerle and Henkel, Ann. Rev. Immunol. 12:141-79, 1994; Barnes and Karin, New Engl. J. Med. 336, 1066-71, 1997; Baeuerle and Baltimore, Cell 87:13-20, 1996; Grilli et al., NF-kB and Rel: Participants in a multiform transcriptional regulatory system (Academic Press, Inc., 1993), vol. 143; Baichwal and Baeuerle, Curr. Biol. 7:94-96, 1997). The prototype member of the family, NFxcexaB, is composed of a dimer of p50 NFxcexaB and p65 RelA (Baeuerle and Baltimore, Cell 53:211-17, 1988; Baeuerle and Baltimore, Genes Dev. 3:1689-98, 1989). NF-xcexaB plays a pivotal role in the highly specific pattern of gene expression observed for immune, inflammatory and acute phase response genes, including interleukin 1, interleukin 8, tumor necrosis factor and certain cell adhesion molecules.
Like other members of the Rel family of transcriptional activators, NF-xcexaB is sequestered in an inactive form in the cytoplasm of most cell types. A variety of extracellular stimuli including mitogens, cytokines, antigens, stress inducing agents, UV light and viral proteins initiate a signal transduction pathway that ultimately leads to NF-xcexaB release and activation. Thus, inhibitors and activators of the signal transduction pathway may be used to alter the level of active NF-xcexaB, and have potential utility in the treatment of diseases associated with NF-xcexaB activation.
Activation of NFxcexaB in response to each of these stimuli is controlled by an inhibitory subunit, IxcexaB, which retains NFxcexaB in the cytoplasm. IxcexaB proteins, of which there are six known members, each contain 5-7 ankyrin-like repeats required for association with the NFxcexaB/Rel dimer and for inhibitory activity (see Beg et al., Genes Dev. 7, 2064-70, 1993; Gilmore and Morin, Trends Genet. 9, 427-33, 1993; Diaz-Meco et al., Mol. Cell. Biol. 13:4770-75, 1993; Haskill et al., Cell 65:1281-89, 1991). IxcexaB proteins include IxcexaBxcex1 and IxcexaBxcex2.
NFxcexaB activation involves the sequential phosphorylation, ubiquitination, and degradation of IxcexaB. Phosphorylation of IxcexaB is highly specific for target residues. For example, phosphorylation of the IxcexaB protein IxcexaBxcex1 takes place at serine residues S32 and S36, and phosphorylation of IxcexaBxcex2 occurs at serine residues S19 and S23. The choreographed series of modification and degradation steps results in nuclear import of transcriptionally active NFxcexaB due to the exposure of a nuclear localization signal on NFxcexaB that was previously masked by IxcexaB (Beg et al., Genes Dev. 6:1899-1913, 1992). Thus, NFxcexaB activation is mediated by a signal transduction cascade that includes one or more specific IxcexaB kinases, a linked series of E1, E2 and E3 ubiquitin enzymes, the 26S proteasome, and the nuclear import machinery. The phosphorylation of IxcexaB is a critical step in NF-xcexaB activation, and the identification of an IxcexaB kinase, as well as proteins that modulate its kinase activity, would further the understanding of the activation process, as well as the development of therapeutic methods.
Several protein kinases have been found to phosphorylate IxcexaB in vitro, including protein kinase A (Ghosh and Baltimore, Nature 344:678-82, 1990), protein kinase C (Ghosh and Baltimore, Nature 344:678-82, 1990) and double stranded RNA-dependent protein kinase (Kumar et al., Proc. Natl. Acad. Sci. USA 91:6288-92, 1994). Constitutive phosphorylation of IxcexaBxcex1 by casein kinase II has also been observed (see Barroga et al., Proc. Natl. Acad. Sci. USA 92:7637-41, 1995). None of these kinases, however appear to be responsible for in vivo activation of NF-xcexaB. For example, phosphorylation of IxcexaBxcex1 in vitro by protein kinase A and protein kinase C prevent its association with NF-xcexaB, and phosphorylation by double-stranded RNA-dependent protein kinase results in dissociation of NF-xcexaB. Neither of these conform to the effect of phosphorylation in vivo, where IxcexaBxcex1 phosphorylation at S32 and S36 does not result in dissociation from NF-xcexaB.
Other previously unknown proteins with IxcexaB kinase activity have been reported, but these proteins also do not appear to be significant activators in vivo. A putative IxcexaBxcex1 kinase was identified by Kuno et al., J. Biol. Chem. 270:27914-27919, 1995, but that kinase appears to phosphorylate residues in the C-terminal region of IxcexaBxcex1, rather than the S32 and S36 residues known to be important for in vivo regulation. Diaz-Meco et al., EMBO J. 13:2842-2848, 1994 also identified a 50 kD IxcexaB kinase, with uncharacterized phosphorylation sites. Schouten et al., EMBO J. 16:3133-44, 1997 identified p90rski as a putative IxcexaBxcex1 kinase; however, p90rski is only activated by TPA and phosphorylates IxcexaBxcex1 only on Ser32, which is insufficient to render IxcexaBxcex1 a target for ubiquitination. Finally, Chen et al, Cell 84:853-862, 1996 identified a kinase that phosphorylates IxcexaBxcex1, but that kinase was identified using a non-physiological inducer of IxcexaBxcex1 kinase activity and requires the addition of exogenous factors for in vitro phosphorylation.
Accordingly, there is a need in the art for an IxcexaB kinase that possesses the substrate specificity and other properties of the in vivo kinase. There is also a need for improved methods for modulating the activity of proteins involved in activation of NF-xcexaand for treating diseases associated with NF-xcexacB activation. The present invention fulfills these needs and further provides other related advantages.
Briefly stated, the present invention provides compositions and methods employing a large, multi-subunit IKK signalsome, or a component or variant thereof. In one aspect, the present invention provides an IKK signalsome capable of specifically phosphorylating IxcexaBxcex1 at residues S32 and S36, and IxcexaKxcex2 at residues 19 and 23, without the addition of exogenous cofactors.
In a further related aspect, a polypeptide comprising a component of an IKK signalsome, or a variant of such a component, is provided, wherein the component has a sequence recited in SEQ ID NO:9. An isolated DNA molecule and recombinant expression vector encoding such a polypeptide, as well as a transfected host cell, are also provided.
In another aspect, methods for preparing an IKK signalsome are provided, comprising combining components of an IKK signalsome in a suitable buffer.
In yet another aspect, methods are provided for phosphorylating a substrate of an IKK signalsome, comprising contacting a substrate with an IKK signalsome or a component thereof, and thereby phosphorylating the substrate.
In a further aspect, the present invention provides a method for screening for an agent that modulates IKK signalsome activity, comprising: (a) contacting a candidate agent with an IKK signalsome, wherein the step of contacting is carried out under conditions and for a time sufficient to allow the candidate agent and the IKK signalsome to interact; and (b) subsequently measuring the ability of the candidate agent to modulate IKK signalsome activity.
Within a related aspect, the present invention provides methods for screening for an agent that modulates IKK signalsome activity, comprising: (a) contacting a candidate agent with a polypeptide comprising a component of an IKK signalsome as described above, wherein the step of contacting is carried out under conditions and for a time sufficient to allow the candidate agent and the polypeptide to interact; and (b) subsequently measuring the ability of the candidate agent to modulate the ability of the polypeptide to phosphorylate an IxcexaB protein.
In another aspect, an antibody is provided that binds to a component (e.g., IKK-1 and/or IKK-2) of an IKK signalsome, where the component is capable of phosphorylating IxcexaBxcex1.
In further aspects, the present invention provides methods for modulating NF-xcexaB activity in a patient, comprising administering to a patient an agent that modulates IxcexaB kinase activity in combination with a pharmaceutically acceptable carrier. Methods are also provided for treating a patient afflicted with a disorder associated with the activation of IKK signalsome, comprising administering to a patient a therapeutically effective amount of an agent that modulates IxcexaB kinase activity in combination with a pharmaceutically acceptable carrier.
In yet another aspect, a method for detecting IKK signalsome activity in a sample is provided, comprising: (a) contacting a sample with an antibody that binds to an IKK signalsome under conditions and for a time sufficient to allow the antibody to immunoprecipitate an IKK signalsome; (b) separating immunoprecipitated material from the sample; and (c) determining the ability of the immunoprecipitated material to specifically phosphorylate an IxcexaB protein with in vivo specificity. Within one such embodiment, the ability of the immunoprecipitated material to phosphorylate IxcexaBxcex1 at residues S32 and/or S36 is determined.
In a related aspect, a kit for detecting IKK signalsome activity in a sample is provided, comprising an antibody that binds to an IKK signalsome in combination with a suitable buffer.
In a further aspect, the present invention provides a method for identifying an upstream kinase in the NF-xcexaB signal transduction cascade, comprising evaluating the ability of a candidate upstream kinase to phosphorylate an IKK signalsome, a component thereof or a variant of such a component.
A method for identifying a component of an IKK signalsome is also provided, comprising: (a) isolating an IKK signalsome; (b) separating the signalsome into components, and (c) obtaining a partial sequence of a component.
In yet another aspect, a method is provided for preparing an IKK signalsome from a biological sample, comprising: (a) separating a biological sample into two or more fractions; and (b) monitoring IxcexaB kinase activity in the fractions.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.